User:Marcin Jozef Suskiewicz/Sandbox Parvin/

Alpha-parvin, also known as actopaxin or CH domain-containing integrin-linked kinase (ILK)-binding protein (CH-ILK-BP) is an adapter protein known to interact with a number of focal adhesion proteins leading to focal adhesion stabilisation. Knock-out analysis confirmed it to be essential for efficient directional cell migration during embryogenesis in mice. Spatially and temporarily regulated dynamic changes in the phosphorylation status of alpha-parvin at serines 4 and 8 and consequent changes in affinities towards its binding partners (icluding CdGAP, TESK1 and possibly others, e.g. ILK) may be responsible for 1) focal adhesion turnover (disassembly of old adhesions, assembly of new ones) and 2) actin cytoskeleton reorganization, two interrelated processes contributing to cell migration.

Focal adhesions and cell migration
The extracellular matrix (ECM) composed of matrix molecules such as collagens, laminins, proteoglycans and non-matrix proteins including growth factors, provides physical support and mediates bidirectional flow of signals in all tissues and organs. Cell-ECM attachments, critical for appropriate localization and functioning of cells, are mediated by integrins. The engagement of integrins to the ECM results in the recruitment of various proteins to the cell periphery and consequent assembly of dot-like focal complexes (FXs or nascent adhesions), which can mature to much larger focal adhesions (FAs or focal contacts). Focal adhesions are bound to the integrins on one hand and to the actin cytoskeleton on the other, thus accounting for the mechanical continuity and signal transduction between the ECM outside the cell and the cytoskeleton inside the cell. The complex FA network (the adhesome), is composed of, among others, actin regulators, Tyr and Ser/Thr kinases and phosphatases, phosphatidylinositols and their modifiers, Rho family GTPases and their effectors. Interactions between these components are mediated by various adapter proteins, including talin, vinculin, paxillin and alpha-parvin. FAs can grow and shrink and, even in stationary cells, disappear on average tens of minutes after maturation. The spatially and temporarily regulated FA turnover together with changes in cytoskeletal contractability enable cell migration in development and regeneration.

Alpha-parvin's interactions in the adhesome
Alpha-parvin is thus a component of the adhesome, but a more basic unit of which it is part is the IPP (ILK-PINCH-parvin) complex. The central component of this complex, ILK, binds both PINCH and alpha-parvin, which do not interact directly with each other, and it is by means of ILK's pseudokinase domain that the complex is recruited to the cytoplasmic tail of integrins. The IPP complex is important for the correct FA assembly and promotes anti-apoptotic signalling. Alpha-parvin is also known to interact with a number of other proteins, including F-actin, paxillin , paxillin's homologue HIC5 , TESK1 , CdGAP and alphaPIX.

Alpha-parvin phosphorylation and cell migration
Alpha-parvin possesses 6 putative proline-directed serine/threonine phosphorylation targets (residues 4, 8, 14, 16, 19, 61), of which serines 4 and 8 were shown to be the most important. Phosphorylation of alpha-parvin at serines 4 and 8 is correlated with the tightly regulated process of FA turnover during cell migration. Firstly, phosphorylation of these residues by cyclin B1/cdc2 is observed in the context of mitosis, whereby it contributes to FA disassembly required for cell-rounding prior to cell division, suggesting it may cause a similar effect during cell migration. Indeed, in migrating cells these residues are observed to be phosphorylated, likely as a result of MAP kinase and/or PI3K. Secondly, phosphomimetic mutations of serines 4 and 8 to aspartates result in faster migration and spreading, while mutations preventing phosphorylation impair these processes. Finally, as has already been mentioned in the introduction, knock-out mice phenotype (embryonic lethality due to severe cardiovascular defects) suggests alpha-parvin deficiency results in impaired directional migration of endothelial cells during embryonic development, heart development in particular. The macroscopic effects of alpha-parvin phosphorylation likely result from the altered affinity for its binding partners. So far it has been demonstrated that phosphorylation at serines 4 and 8 affects binding of alpha-parvin to TESK1 and CdGAP. When TESK1 is bound to alpha-parvin it is prevented from severing actin fibres. TESK1 is thought to be released from inhibition upon alpha-parvin phosphorylation and this can contribute to the decomposition of actin fibres and FA disassembly. CdGAP, on the other hand, is involved in the regulation of small GTPase signalling, which accounts for changes in cytoskeletal contractability during cell migration. Evidence suggesting that also the interaction with ILK is affected by phosphorylation is not strong, but it is likely that this or yet other binding partners bind in the phosphorylation-dependent manner.

Domain composition
Alpha-parvin's structure can be divided into four regions: 1) N-terminal flexible domain (residues 1-96), 2) N-terminal CH domain (97-200 according to SMART ), 3) linker region (201-241) and 4) C-terminal CH domain (242-372 identified by limited subtilisin proteolysis ). Most interactions of alpha-parvin are mapped to the C-terminal CH domain, but CdGAP and perhaps alphaPIX or other, yet unknown partners, interact with the N-terminal flexibile domain. This flexible domain seems to lack a well defined 3D structure and can therefore be classified as a putative intrinsically disordered region. The interactions of this regions with the binding partners are therefore likely to be characterized by relatively low affinity, but high affinity nonetheless. The abovementioned phosphorylation sites (serines 4 and 8) involved in focal adhesion regulation are located in this segment, which makes them so called disorder-enhanced phosphorylation sites.

C-terminal CH domain
The calponin-homology (CH) domains are helical structural units around 100 amino acids long. They comprise at least four helices, three of them forming a helical bundle. CH domains usually comprise elements of big multidomain proteins and are present either in singlet or duplex/tandem arrangement. The tandem arrangement of CH domains is often associated with F-actin binding (and is thus called actin-binding domain or ABD), but generally CH domains seem to be characterized by functional plasticity and ability to bind various structural motifs. In the case of alpha-parvin, the interactions of CH domains with both F-actin and other partners (paxillin, ILK) are observed. The interactions with paxillin and ILK are mediated by a single CH domain, the C-terminal one. This domain has attracted most attention. While no full-length structure of alpha-parvin has been solved to date, the structure of the C-terminal CH domain, on its own and in complexes (with paxillin and the pseudokinase domain of ILK ) are available.

 2vzc shows the structure of the C-terminal CH domain of alpha-parvin at around 1.05 Å. When a sequence alignment of alpha-parvin with all its sequence homologues is performed and the protein is labeled according to the degree of sequence conservation, one can see that the highest conservation is exhibited by the residues located in the core helices , and less is seen in linker helices and loops. When the structural superposition of the C-terminal CH domain of alpha-parvin and one of the CH domains of alpha-actinin 3 (1vku) is performed, a good overlap is observed despite low sequence homology (≤26% identity), as represented by the RMSD of 1.19 Å for 103 equivalent Cα positions. This suggests that the structural framework of CH domain is quite robust. The most diverged fragments in the C-terminal CH domain of alpha-parvin correspond to 1) an additional helix (so called N-terminal linker helix) located at the N-terminal end of the domain and not observed in any other CH domain and 2) a long loop between two of the helices. The loop in question contains a 3-amino acid insertion (313-315). 